Project Summary (Abstract) Globally, there are nearly 1.7 billion cases of enteric GI infections that cause diarrheal disease every year. More disturbing, is that diarrheal disease caused by enteric pathogens kills over 2,000 children every day and is the second leading cause of death among children under the age of five. Enteropathogenic Escherichia coli (EPEC) is a common culprit of diarrheal disease in Sub-Saharan Africa, accounting for roughly 10% of all diarrheal deaths. EPEC infections are disproportionately high in children with HIV and cause more frequent and prolonged diarrhea episodes than in the uninfected counterparts. Despite the immune deficiency caused by HIV infection, EPEC is the only enteric pathogen that is frequently and consistently prevalent in this population, suggesting that more than just immune suppression may be involved in this relationship. Further, we have found that HIV uninfected children receiving the antibiotic cotrimoxazole (CTX) also have higher susceptibility and severity of EPEC infection. This is especially intriguing as both antibiotic use and HIV-infection have been correlated with a reduction in gut microbial diversity and has prompted us to hypothesize that the susceptibility and clinical severity of EPEC infection observed in HIV-infected and antibiotic-treated children is a consequence of a microbiome reduced in diversity, which promotes changes in the metabolic and virulence gene expression by EPEC. We will test this in two aims. In Aim 1 we will assess the microbiome as a potential modulator of EPEC virulence by performing 16S sequencing on HIV+ and antibiotic-treated children to confirm reduced diversity and to compare bacterial compositions, we will examine the association between EPEC strain and clinical outcomes and we will use germ-free mice reconstituted with human stool to evaluate whether changes in diversity impact EPEC infection. In Aim 2, we will determine genetic alterations in the clinical isolates using microbial transcriptomics and then we will determine whether the genetic data is correct by assessing virulence in an animal model and we will determine whether restoring microbial diversity via a fecal microbiota transplant would be an effective treatment option.